Pentachloronitrobenzene (PCNB) has been widely utilized as a fungicide to control diseases. However, toxic effect data of PCNB on terrestrial invertebrate are not available till now. Herein, the earthworms (Eisenia fetida) were exposed to soil containing different levels of PCNB. Mortality, weight, accumulation, and physiological indexes of earthworms were determined on certain days. PCNB inhibited the growth of earthworms and induced a significant increase in the activity of antioxidative enzymes. ROS, SOD, and MDA of earthworms in the highest treatment group were 6.8, 4.4, and 3.8 times higher than those in the control group, respectively. In addition, earthworm coelomocytes were successfully extracted, cultured, and innovatively employed in in-vitro toxicity test to evaluate the toxic effect of PCNB. The biomarkers utilized in in-vitro toxicity test, including cell viability, intracellular ROS and extracellular LDH showed significant correlations with the PCNB in the culture media, indicating that the in-vitro toxicity test may serve as a useful tool for toxic assessment of pollutants to earthworms and other organisms.